3D reconstruction showing three types of nanofilaments that connect to synaptic vesicles in the nerve terminals of excitatory synapses in the rat hippocampus. Reprinted with permission: Cole, A. A., et al., Journal of Neuroscience (2016).

This spectacular image – which took the best part of a year to create – shows the fine structure of a nerve terminal at high resolution, revealing, for the very first time, an intricate network of fine filaments that controls the movements of synaptic vesicles.

The brain is soft and wet, with the consistency of a lump of jelly. Yet, it is the most complex and highly organized structure that we know of, containing hundreds of billions of neurons and glial cells, and something on the order of one quadrillion synaptic connections, all of which are arranged in a very specific manner.

This high degree of specificity extends down to the deepest levels of brain organization. Just beneath the membrane at the nerve terminal, synaptic vesicles store neurotransmitter molecules, and await the arrival of a nervous impulse, whereupon they fuse with the membrane and release their contents into the synaptic cleft, the miniscule gap at the junction between nerve cells, and diffuse across it to bind to receptor protein molecules embedded at the surface of the partner cell.

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The process of neurotransmitter release is tightly orchestrated. Ready vesicles are ‘docked’ in the ‘active zone’ lying beneath the cell membrane, and are depleted when they fuse with the membrane, only to be replenished from a reservoir of pre-prepared vesicles located further inside the cell. Spent vesicles are quickly pulled back out of the membrane, reformed, refilled with neurotransmitter molecules, and then returned to the reservoir, so that they can be shuttled into the active zone when needed. An individual nerve cell may use up hundreds, or perhaps thousands, of vesicles every second, and so this recycling process occurs continuously to maintain the signalling between nerve cells.

The nerve terminal contains more than 400 different proteins, which together form the exquisite molecular machinery that regulates the fusion, recycling, and movements of synaptic vesicles between the reservoir pool, active zone and cell membrane. Although modern molecular methods have revealed a great deal about the identity and function of many of these proteins, we still know very little about how they are organised at the nerve terminal, because the structures they form are extremely fragile, and researchers lacked appropriate ways of studying them.

One way of looking at the fine structure of synapses is electron microscopy. This technique, developed in the 1930s, enabled researchers to examine synapses and other neuronal structures in unprecedented detail, but can only capture images at the surface of the specimen being studied. Recent advances in this method now make it possible to access and visualise structures that lie deeper inside synapses.

Andy Cole of the National Institute of Neurological Disorders and Stroke (NINDS) in Bethesda, Maryland and his colleagues exploited these advances to produce three-dimensional reconstructions of the entire cloud of synaptic vesicles lying within 250 nanometers (nm, or billionths of a meter) of the active zone in excitatory synapses of hippocampal neurons dissected from the brains of adult rats.

“There are two challenges when dealing with fine structures: preserving the structure and extracting the details,” says Cole. “We freeze the specimen in milliseconds, at very low temperature and very high pressure, which gives us a snapshot in time of the synapse with little to no distortion. Then, we embed the specimen in plastic, so we can shoot the specimen with a very high-energy electron beam without destroying the structure.”

“To extract more detail from our specimen, we take over a hundred images from different angles, producing a block of data rather than one 2D image,” he adds, explaining that the difference between this method and conventional electron microscopy “is analogous to the difference between an X-Ray and an MRI”.

Their results, published recently in the Journal of Neuroscience, show that three different types of filaments come into contact with synaptic vesicles at the nerve terminal, distinguished primarily by their shape and length. One type appears to be straight, with an average of length 22nm, and has a spherical base that connects it to the membrane in the active zone. These are ‘docking’ filaments, each of which contacts a single vesicle, and keeps it in place so that it is ready to fuse with the membrane (shown in green in the image above).

A second type is slightly longer and also straight, but is somewhat thicker and lacks the spherical base; these ‘bridge’ filaments connect pairs of docked vesicles together in the active zone, and make up nearly two thirds of the total population of filaments seen (shown in purple). The third type is longer still, and characterised by numerous kins and branches; these ‘cluster’ filaments (rendered in gold and white) connect multiple vesicles to each other and also to the membrane.

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The process of collecting these data and rendering them to produce the reconstructions is extremely laborious and time-consuming. “From the point of having a data set in hand to having a finished rendering, it can take as long as a year,” says Cole, “[because] making sense of the renderings takes a few months and another data set at least. Limiting the number of rendered objects speeds things up, which is why we chose to limit how deep into the presynaptic terminal we rendered.”

Although the role of each type of filament can be inferred from its structure and positioning, the exact function of each is still not entirely clear. The molecular composition of the filaments – the identity of the proteins that make up each one – also remains to be determined. It’s also important to remember that there are many different kinds of synapses in the brain: Cole and his colleagues examined nerve terminals in the rat hippocampus that release glutamate, but terminals in other parts of the brain, and those which release other neurotransmitters, could well be organised differently.

What’s more, all of these structures are moveable parts, and the method used captures just a snapshot in time, but further advances in techniques such as super-resolution microscopy may eventually enable researchers to watch them in action in live cells. “We have many more data sets waiting for analysis, and I am sure there are many more discoveries to be made by those with the patience to look,” says Cole.